Regulation of AU-rich element-mediated mRNA decay

描述(由申请人提供)：信使核糖核酸稳定性的调节在基因表达的控制中起着关键作用。在多种病理过程中都涉及到mRNA的异常转换。了解信使核糖核酸的衰变规律，有助于深入了解某些疾病的发病机制。哺乳动物细胞中一个重要的顺式作用元件是富含AU的元件(ARE)，它通过ARE介导的mRNA衰变(AMD)过程来指导mRNA衰变。在过去的七年里，我们研究了KSRP在AMD中的功能，KSRP是一种促进衰老的ARE结合蛋白。为了进一步了解KSRP在AMD中的活性调节，并确定其在体内的生理mRNA靶点，我们启动了一项纯化计划，以分离与KSRP相关的蛋白并产生KSRP缺失的小鼠。此次续签赠款将利用一系列生化和分子方法来实现以下具体目标。目的1研究与KSRP共纯化的RNA解旋酶DDX1在调节AMD和KSRP亚细胞定位中的作用。这一目标还将大规模纯化KSRP复合体，通过质谱学鉴定与KSRP共纯化的蛋白质，并研究它们在AMD调节中的作用。具体目的2是利用KSRP基因敲除小鼠，研究KSRP在I型干扰素(干扰素-1和干扰素-2)基因转录后调控和病毒感染中的作用。这一目的将检验KSRP是否调节IFNA和Ifnb mRNAs的衰退，以及KSRP缺乏对病毒感染的影响。特异性目标3是在内毒素刺激的巨噬细胞中识别KSRP靶向的mRNAs。我们将通过全基因组的mRNA衰变分析，以及通过核糖核蛋白免疫沉淀和微阵列分析，鉴定在内毒素刺激的KSRP-/-巨噬细胞中稳定的mRNAs和与KSRP相关的mRNA。我们的长期目标是了解KSRP调控AMD的机制，识别其体内的mRNA靶标，并表征与靶标调节失调相关的表型。KSRP基因敲除小鼠的建立为研究KSRP对某些细胞因子mRNAs转录后调控提供了有价值的工具，这将有助于我们理解由于mRNA衰退缺陷而导致的炎症性和免疫性疾病的发病机制。 与公共卫生相关：信使RNA(信使核糖核酸)稳定性的异常调节与各种病理过程有关。了解信使核糖核酸的降解规律将有助于深入了解该病的发病机制。我们的研究应该允许开发免疫和炎症性疾病以及癌症的替代治疗方法。

DESCRIPTION (provided by applicant): Regulation of mRNA stability plays a critical role in the control of gene expression. Dysregulated mRNA turnover has been implicated in various pathological processes. Understanding the regulation of mRNA decay should provide insights into the pathogenesis of certain disease. An important cis-acting element responsible for rapid mRNA decay in mammalian cells is the AU-rich element (ARE), which directs mRNA decay through a process referred to as ARE-mediated mRNA decay (AMD). We have investigated the function of KSRP, a decay-promoting ARE-binding protein, in AMD for the past seven years. To further understand the regulation of KSRP activity in AMD and identify it's in vivo physiological mRNA targets, we have initiated a purification scheme to isolate proteins that associate with KSRP and have generated Ksrp- null mice. This grant renewal will utilize a series of biochemical and molecular approaches to achieve the following specific aims. Specific aim 1 is to characterize function of DDX1, an RNA helicase that was found to co-purify with KSRP, in regulation of AMD and the subcellular localization of KSRP. This aim will also purify KSRP complexes in a large scale, identify proteins that co-purify with KSRP by mass spectrometry, and investigate their roles in the regulation of AMD. Specific aim 2 is to investigate the roles of Ksrp in post- transcriptional control of type I interferon (IFN-1 and IFN-2) gene expression and in virus infection using Ksrp knockout mice. This aim will examine whether Ksrp regulates the decay of Ifna and Ifnb mRNAs and the effect of Ksrp deficiency on virus infection. Specific aim 3 is to identify Ksrp target mRNAs in LPS- stimulated macrophages. We will identify mRNAs that are stabilized in LPS-stimulated Ksrp-/- macrophages by genome-wide analysis of mRNA decay and those associated with Ksrp by ribonucleoprotein immunoprecipitation and microarray analysis. Our long-term goals are to understand the mechanism by which KSRP regulates AMD, to identify it's in vivo mRNA targets, and to characterize phenotypes associated with the dysregulation of the targets. The generation of Ksrp knockout mice should provide a valuable tool to study the post-transcriptional regulation of certain cytokine mRNAs by Ksrp, which should contribute to our understanding of the pathogenesis of inflammatory and immunological diseases resulting from a defect in mRNA decay. PUBLIC HEALTH RELEVANCE: Aberrant regulation of messenger RNA (mRNA) stability has been implicated in various pathological processes. Understanding the regulation of mRNA degradation would provide insights into the disease pathogenesis. Our research should allow for the development of alternative treatments for immune and inflammatory diseases and cancer.